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Microbial eukaryotes have adapted to hypoxia by horizontal acquisitions of a gene involved in rhodoquinone biosynthesis

  1. Courtney W Stairs
  2. Laura Eme
  3. Sergio A Muñoz-Gómez
  4. Alejandro Cohen
  5. Graham Dellaire
  6. Jennifer N Shepherd
  7. James P Fawcett
  8. Andrew J Roger  Is a corresponding author
  1. Dalhousie University, Canada
  2. Gonzaga University, United States
Research Article
Cite this article as: eLife 2018;7:e34292 doi: 10.7554/eLife.34292
6 figures, 2 tables, 1 data set and 5 additional files

Figures

Structure and function of ubiquinone and rhodoquinone in mitochondria.

(A) Under aerobic conditions, electrons from NADH or succinate (Suc) are shuttled to ubiquinone (UQ) via Complex I (I) or Complex II (II) respectively to generate NAD (NAD+), fumarate (fum) and ubiquinol (UQH2). The electron transfer via Complex I fuels the transport of protons from the mitochondrial matrix into the intermembrane space (IMS). Electrons from UQH2 are transferred to Complex III, cytochrome c, and ultimately O2 via Complex IV with the concomitant pumping of protons. Complex V (V) uses this proton gradient to synthesize ATP. (B) In anaerobic eukaryotes, electrons from NADH are shuttled to rhodoquinone (RQ) to generate rhodoquinol (RH2). The RQ pool is regenerated via CII functioning as a fumarate reductase.

https://doi.org/10.7554/eLife.34292.002
Figure 2 with 3 supplements
Maximum-likelihood phylogeny of RquA proteins constructed from an alignment of homologs from 166 organisms and 197 aligned sites.

Eukaryotic proteins are coloured based on their phylogenetic affiliations: Obazoa (purple), Stramenopiles-Alveolata (orange), Excavata (green), Amoebozoa (magenta) and Rhizaria (blue). Hexagons represent taxa where RQ has been detected experimentally. Proteobacterial designations (α,β,γ) are indicated in the grey squares. Genetic linkage of rquA and genes related to respiratory function (complex I-IV, cytochrome c metabolism, or heme metabolism) are shown with chain-links and detailed in Supplementary file 1. When these indications are in a collapsed node, the number of genomes showing linkage are shown in brackets. Bootstrap values (or posterior probability) greater than 70 (0.7) and 90 (0.9) are shown with open circles (squares) or closed circles (squares) respectively. The presence of spliceosomal introns in the eukaryotic sequences are indicated with ‘i' in a box. Dashed branches were made shorter by 50% to facilitate visualization.

https://doi.org/10.7554/eLife.34292.003
Figure 2—figure supplement 1
Intron positions in eukaryotic rquA gene sequences.

Nucleotide from both transcriptome and genomic sequencing projects (when available) were aligned using Sequencher (Gene Codes v 5.4.6) and introns were manually inspected for position, length and phase. The phase indicates the position of the intron relative to the codon where phase 0, 1 and 2 begin before the codon, in between position 1 and 2, or in between position 2 and 3 respectively. All introns were major spliceosomal introns with GT-AG boundaries. In general, intron positions and lengths were not conserved in distantly related species.

https://doi.org/10.7554/eLife.34292.004
Figure 2—figure supplement 2
Phyletic distribution of RQUA among alphaproteobacteria superimposed on the alphaproteobacterial species tree.

Maximum likelihood analysis of a 200 phylum-specific phylogenetic marker genes representing 54,400 sites from 210 representative alphaproteobacteria under the LG + C60 + F (PMSF)+ Γ4 model of evolution implemented in IQ-TREE. Tree was rooted using an outgroup of other proteobacteria. Nodes with maximal support are unlabeled, while those with support values between 95–99 are labeled with squares. RQUA-containing taxa are coloured in purple or orange representing Group A and Group B type RQUA respectively. Orders of alphaproteobacteria are indicated on the right of the tree with a summary of the number of alphaproteobacterial genomes interrogated for RQUA presence/absence in Genbank (green box).

https://doi.org/10.7554/eLife.34292.005
Figure 2—figure supplement 3
RquA homologues lack critical S-adenosyl methionine (SAM) binding site.

Conserved residues known to interact with the carbonyl group of SAM are shown with black triangles. A substitution from aspartate to glutamine was obversed in all bacterial (represented here by Rhodospirillum rubrum) and eukaryotic RquA demonstrated with a open circle. Taxa are coloured with super-Group Affiliation as in Figure 2. Motif features: [VILFG]-[LIVCS]-[DENLV]-[VALMIT]-[GLYCFA]-[CSTAYFPAG]-[GA]-[PTSGNKRMV]-[GD]).

https://doi.org/10.7554/eLife.34292.006
RquA co-occurs with quinone-utilizing enzymes.

Eukaryotic genomes and transcriptomes were surveyed for homologs of respiratory chain components (Complexes I-V, CI-CV), alternative oxidase (AOX), dihydroorotate dehydrogenase (DHOH), electron-transferring flavoprotein system (ETF), glycerol-3-phosphate dehydrogenase (G3P), sulfite:quinone oxidoreductase (SQO), RquA, and one or more anaerobiosis-associated protein (AAP; detailed in Supplementary file 1). Grey and white circles indicate that homologs were not detected in transcriptome and genome sequence data respectively. Half circle in CI for Pygsuia biforma and Trichomonas vaginalis indicates only two subunits (NUOE and NUOF) were identified. ‘ψ ‘indicates pseudogenes.

https://doi.org/10.7554/eLife.34292.011
Figure 4 with 3 supplements
Subcellular localization of RquA and rhodoquinone production in Pygsuia biforma.

(A) Antibodies raised against RquA (green) colocalized with MitoTracker (red). Confocal slices (0.3 μm) were deconvoluted (using a constrained interative algorithm) and combined to render a 3D image. DAPI stained nuclei (blue) were volume rendered in Imaris for clarity. (B) Lipid extracts were separated by liquid chromatography and analyzed with selected-reaction monitoring mass spectrometry. Rhodoquinone species eluted from the column in roughly 3 min intervals as chain length increases (RQ8-10). Diagnostic product ions corresponding to the rhodoquinone head group (182.1 m/z) following fragmentation of parent ions were detected.

https://doi.org/10.7554/eLife.34292.012
Figure 4—figure supplement 1
Antibodies directed against Pygsuia RquA (PbRquA) recognize purified recombinant PbRquA by western blot analysis.

Proteins isolated from whole cell extracts of E. coli induced to express pGEX4T-1 (1) and pGEX- Pb-rquA (2) and glutathione-S-transferase(GST)-Pb-rquA purified with glutathione magnetic beads (3) were resolved by SDS-PAGE and probed by immunoblotting using anti- PbRquA. The estimated molecular weight of the GST-PbRquA is 56 kDa (i.e., 26 kDa GST and 30 kDa for PbRquA). The antibody interacted with an endogenous E. coli protein (1 and 2, lower signal) and this signal was mostly reduced upon purification of the protein (3).

https://doi.org/10.7554/eLife.34292.013
Figure 4—figure supplement 2
Spectra represent the elution times of detection of the rhodoquinone head group (182.1 m/z; left) and ubiquinone head group (197.1 m/z) following fragmentation of parent ions of different isoprenyl chain lengths as indicated.
https://doi.org/10.7554/eLife.34292.014
Figure 4—figure supplement 3
Lipid extracts were separated by high-performance liquid chromatography and analyzed with mixed-reaction monitoring mass spectrometry from Pygsuia mixed culture, Pygsuia ‘food’ bacteria culture, glassware, and Rhodospirillum rubrum as indicated. 

Spectra represent the elution times of detection of the rhodoquinone head group (182.1 m/z; left) and ubiquinone head group (197.1 m/z) following fragmentation of parent ions of different isoprenyl chain lengths as indicated.

https://doi.org/10.7554/eLife.34292.015
The interactions of rhodoquinone with other mitochondrial redox reactions in different eukaryotes with mitochondrion-related organelles.

Standard reduction potentials for each major reaction involved in rhodoquinone (R)) and ubiquinone (U) metabolism are shown in increasing order of potential. Half reaction equations are detailed in supplementary file 1. The electron transfer is more favourable when passed to a species with a more positive standard reduction potential (i.e. from left to right). Abbreviations: I, Complex I; II, Complex II; III, Complex III; IV, Complex IV; V, Complex V; E, electron transferring flavoprotein dehydrogenase; G, glycerol-3-phosphate (G3P) dehydrogenase; A alternative oxidase; Fum, fumarate; Suc, succinate; DHAP, dihydroxyacetone phosphate; and Cyt c, cytrochrome c. U* Indicates that the involvement of ubiquinone is unknown, ? indicates the direction of electron transfer is unknown. Absence of a circle indicates that no homologs were detected in the organism. Genes undergoing pseudogenization are shown in white cirlces.

https://doi.org/10.7554/eLife.34292.016
Author response image 1
Cumulative frequency plot of transcriptome completeness for the marine microbial eukaryotes sequencing projects.
https://doi.org/10.7554/eLife.34292.023

Tables

Table 1
Approximate unbiased topology tests for RquA analyses.
https://doi.org/10.7554/eLife.34292.007
Monophyly TestedTree fileCONSEL p-AUa
Group A and Group B
Maximum likelihood treeFigure 2; Supplementary file 2 - tree 20.743
Group A eukaryotes + Group B
eukaryotes
Supplementary file 2 - tree 33.00E-37***
Group A1 eukaryotes: Blastocystis, Proteromonas,
Neoparamoebids, Euglenids, Pygsuia
Supplementary file 2 - tree 40.622
Group A1 eukaryotes + BrevimastigamonasSupplementary file 2 - tree 50.46
Group A1 eukaryotes + Brevimastigamonas
+ Mastigamoeba
Supplementary file 2 - tree 60.294
Group A eukaryotesSupplementary file 2 - tree 70.253
Group B eukaryotesSupplementary file 2 - tree 80.179
Obazoa (Pygsuia + Monosiga)Supplementary file 2 - tree 90.002**
Amoerphea (Obazoa + Amoebozoa)Supplementary file 2 - tree 101.00E-32***
AmoebozoaSupplementary file 2 - tree 110.206
Stramenopiles + AlveolatesSupplementary file 2 - tree 120.004**
Stramenopiles + Alveolates +
Rhizaria (SAR)
Supplementary file 2 - tree 130.034*
Diaphoretickes
(SAR + Euglenids)
Supplementary file 2 - tree 140.018*
RhizariaSupplementary file 2 - tree 151.00E-60***
Eukaryotes + MAG
alphaproteobacteria
Supplementary file 2 - tree 162.00E-41***
Group A eukaryotes + MAG
alphaproteobacteria
Supplementary file 2 - tree 170.227
AlphaproteobacteriaSupplementary file 2 - tree 185.00E-34***
Eukaryotes + alphaproteobacteriaSupplementary file 2 - tree 198.00E-43***
Group A eukaryotes +
Group A alphaproteobacteria
Supplementary file 2 - tree 203.00E-31***
Group B eukaryotes +
Group B alphaproteobacteria
Supplementary file 2 - tree 214.00E-05***
Group A
Maximum likelihood treeSupplementary file 3 - tree 10.892
EukaryotesSupplementary file 3 - tree 20.225
AmoebozoaSupplementary file 3 - tree 30.315
Pygsuia + Amoebozoa (Amorphea)Supplementary file 3 - tree 40.22
Eukaryotes + alphaproteobacteriaSupplementary file 3 - tree 53.00E-59***
Eukaryotes + MAG
alphaproteobacteria
Supplementary file 3 - tree 60.226
Group B
Maximum likelihood treeSupplementary file 4 - tree 10.827
EukaryotesSupplementary file 4 - tree 20.081
Stramenopiles + AlveolatesSupplementary file 4 - tree 30.287
SARSupplementary file 4 - tree 40.281
Eukaryotes + alphaproteobacteriaSupplementary file 4 - tree 52.00E-75***
  1. aitalicized values indicate topologies that could not be rejected (p<0.05).

    * 0.05 > p > 0.01

  2. ** 0.01 > p > 0.001

    *** p < 0.001

Table 1—source data 1

Topology test output from CONSEL.

Trees 1-20 represent trees 2-21 from Supplementary file 2; trees 21-120 represent 100 bootstrap trees from the maximum likelihood analysis. Relevant column headers: Obs, observed log-likelihood value; au, topology test p-value; np, bootstrap probability. Details on the column headers can be found at http://stat.sys.i.kyoto-u.ac.jp/prog/consel/quick.html

https://doi.org/10.7554/eLife.34292.008
Table 1—source data 2

Topology test output from CONSEL.

Trees 1-6 represent trees1-6 from Supplementary file 3; trees 7-106 represent 100 bootstrap trees from the maximum likelihood analysis. Relevant column headers: Obs, observed log-likelihood value; au, topology test p-value; np, bootstrap probability. Details on the column headers can be found at http://stat.sys.i.kyoto-u.ac.jp/prog/consel/quick.html

https://doi.org/10.7554/eLife.34292.009
Table 1—source data 3

Topology test output from CONSEL.

Trees 1-5 represent trees 1-5 from Supplementary file 4; trees 6-105 represent 100 bootstrap trees from the maximum likelihood analysis. Relevant column headers: Obs, observed log-likelihood value; au, topology test p-value; np, bootstrap probability. Details on the column headers can be found at http://stat.sys.i.kyoto-u.ac.jp/prog/consel/quick.html

https://doi.org/10.7554/eLife.34292.010
Author response table 1
Numbers of complete genomes and transcriptome projects surveyed from each of the major lineage of eukaryotes.
https://doi.org/10.7554/eLife.34292.024

RquA detecteda

NCBI Genomesb

NCIB TSA nrc

MMETSPd

Alveolata710532134
Amoebozoa441108
Apusozoa0100
Breviatea1110
Centroheliozoa0010
Cryptophyta08125
Euglenozoa

2e

5535
Fornicata0220
Glaucocystophyceae0403
Haptophyceae05261
Heterolobosea0512
Jakobida0600
Malawimonadidae0200
Opisthokonta19,240139510
Oxymonadida0110
Parabasalia0210
Rhizaria412421
Rhodophyta0124158
Stramenopiles515049279
Viridiplantae02,46149972
unclassified00319
Multispecies with undefined taxa ID008219
Total24122252102647
a Source: this study
b Source: NCBI Taxonomy Browser, selecting “Genomes” – January 28, 2018

c Source NCBI Trace Archive for the transcriptome shotgun assembly database availble at https://www.ncbi.nlm.nih.gov/Traces/wgs/?term=tsa Taxonomy ID were extracted and taxonomy parsed with ete-toolkit. Only non-redundant taxonomy IDs are shown.

d Source: iMicrobe; Taxonomy ID were extracted and taxonomy assigned with ete-toolkit. Only non-redundant taxonomy IDs are shown.
e Three copies were identified in Eutriptiella and 1 in Euglena

Data availability

All data is available on Dryad DOI: https://doi.org/10.5061/dryad.qp745

The following data sets were generated
  1. 1

Additional files

Supplementary file 1

Excel file with workbooks containing information about the number of genomes surveyed, PFAM domains of putative RquA homologues, gene accession numbers for RquA and Q-utilizing proteins, mitochondrial targeting sequence information, redox half-potentials used for Figure 5, bacterial genes with and without linkage to respiratory complexes.

https://doi.org/10.7554/eLife.34292.017
Supplementary file 2

PDF with all the trees for the full RquA phylogenetic analysis and associated topology tests

https://doi.org/10.7554/eLife.34292.018
Supplementary file 3

PDF with all the tree for the Group A phylogenetic analysis and associated topology tests

https://doi.org/10.7554/eLife.34292.019
Supplementary file 4

PDF with all the tree for the Group B phylogenetic analysis and associated topology tests

https://doi.org/10.7554/eLife.34292.020
Transparent reporting form
https://doi.org/10.7554/eLife.34292.021

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